The present invention is in the field of inhibitors of HMG-CoA reductase which are useful as antihypercholesterolemic agents. It is now well established that hypercholesterolemia is a significant risk factor in the development of cardiovascular disease, particularly atherosclerosis. Compounds which are able to inhibit the HMG-CoA reductase enzyme interfere with and limit the biosynthesis of cholesterol, and in that way function as antihypercholesterolemic agents. Such compounds, especially the natural fermentation products compactin and mevinolin, are now well known. There is a continuous search, nevertheless, for additional analogs which will give improved antihypercholesterolemic performance. The triol acid produced by enzymatic hydrolysis of lovastatin acid using an enzyme derived from Clonostachys compactiuscula in accordance with the biosynthetic process of the present invention provides quantities of a starting material for the preparation and production of such semisynthetic analogs. ##STR1##
The process of this invention may also be conducted starting with pravastatin, which differs from lovastatin in that the 6-.alpha.-methyl group on the hexahydronaphthyl ring is replaced with a 6-.beta.-hydroxyl group. Treatment of pravastatin with Clonostachys compactiuscula in accordance with the biosynthetic process of the present invention provides the corresponding pravastatin triol acid below. ##STR2##
As already described above, the triol acid and its lactone form are old compounds. The triol acid in its lactone form, for example, is described in Endo, published Japanese Pat. Appln. 86-13798 (1986), where its production by fermentation of Monascus ruber and a demonstration of its ability to reduce blood cholesterol levels is also set out. The triol acid in its lactone form, as well as the triol acid itself, are also described in Willard U.S. Pat. No. 4,293,496 (1981). However, in Willard, these compounds are prepared by chemical hydrolysis to remove the 8-(.alpha.-methylbutyryloxy) ester side chain of lovastatin, the starting material which is a fermentation product of a particular strain of Aspergillus terreus. There is no suggestion that such hydrolysis might be carried out biochemically or microbiologically.
Lovastatin and simvastatin are also compounds known in the art as HMG-CoA reductase inhibitors. The two compounds differ in that lovastatin has a 2-methylbutyryloxy side chain at the 8'-position and simvastatin has a 2,2-dimethylbutyryloxy side chain. ##STR3##
Although simvastatin has been formed from lovastatin, it has been difficult to separate and purify simvastatin from a mixture of simvastatin and lovastatin. The similarity in structure between the two compounds (the two compounds differ by only one methyl group) makes high pressure liquid chromatography (HPLC) separation difficult because the compounds have such similar retention times. One methodology used to isolate simvastatin from a mixture of simvastatin and lovastatin is to convert the unreacted lovastatin to the triol acid or the diol lactone using base hydrolysis with, for example, sodium hydroxide (NaOH) or lithium hydroxide (LiOH). However, this base hydrolysis hydrolyzes only a percentage of the lovastatin, leaving unreacted lovastatin as a contaminant of the final simvastatin product. An additional problem with the base hydrolysis is partial hydrolysis of the simvastatin, thus reducing the yield of the desired simvastatin product. The present invention provides for a process of isolating simvastatin from mixtures of simvastatin and lovastatin in greater purity and without concomitant yield losses.
Komagata et al., J. Antibiotics, 39, 1574-77 (1986), describes enzymatic hydrolytic conversion of compactin (ML-236B) to the 8-hydroxy analog (ML-236A) in which the same side chain is removed as in the present invention. Of 1600 fungal strains investigated, 59 were found to be effective in catalyzing the hydrolytic reaction, and Emericella unguis showed the most potent activity. However, C. compactiuscula is not disclosed.
Endo, published Japanese Pat. Appln. 85-176595 (1985) describes the same conversion as Komagata et al. above, but additionally includes conversion of "monacolin K" (which is lovastatin) to "monacolin J", (which is the triol acid in the present invention). Especially useful are said to be the molds Mortierella isabellina, Emericella unguis, Diheterospora chlamydosporia, Humicola fuscoatra, Dichotomomyces cejpii, Neocosmospora africana, Xylogone sphaerospora, Torulomyces ragena, and Thielavia fimeti. However, the highest conversion rate is 78% for a starting material concentration of 0.5 mg/ml, compared to 90-100% with the present invention. And, there is an indication in the related Komagata et al. paper that at higher concentrations, such as the 2.5 mg/ml employed in the present invention, there is a significant drop-off in efficiency of the enzyme. Thus, there is no suggestion in the prior art of the improved microbiological hydrolysis which can be achieved using Clonostachys compactiuscula.
Lovastatin can be converted to a more active HMG-CoA reductase inhibitor by C-methylation of the natural 2(S)-methylbutyryloxy side chain to obtain simvastatin. C-methylation may be accomplished by any known process amenable to the functionalities of the molecule.
One process for direct C-methylation of the 2(S)-methylbutyryloxy side chain is described in U.S. Pat. No. 4,582,915. This process is detailed in Scheme I and in the description which follows. ##STR4## wherein: M is an alkali metal salt, preferably potassium;
X is halo, such as chloro, bromo or iodo, preferably bromo or iodo; PA0 M.sub.1.sup.+ is a cation derived from lithium, sodium or potassium, preferably lithium; and PA0 R.sup.1 and R.sup.2 are
1) independently C.sub.1-3 alkyl, or PA1 2) R.sup.1 and R.sup.2 joined together form a 5- or 6-membered heterocycle such as pyrrolidine or piperidine with the nitrogen to which they are attached, preferably pyrrolidine. PA1 a) H, PA1 b) an alkali metal salt such as Li, Na or K, PA1 c) an alkaline earth metal salt such as Ca or Mg, PA1 d) a salt with other metals such as Al, Fe, Zn, Cu, Ni or Co, PA1 e) an amino acid salt formed from a basic amino acid such as arginine, lysine, .alpha.,.beta.-diaminobutyric acid, or ornithine, PA1 f) an amine salt such as t-octylamine, dibenzylamine, ethylenediamine, morpholine, or tris(hydroxy-methyl) aminomethane, and PA1 g) the ammonium salt.
In the process of forming simvastatin by the direct methylation of lovastatin, the lovastatin lactone compound is first converted to an alkali metal salt, preferably a potassium salt of the dihydroxycarboxylate. Although any conceivable method preparing a dry salt would suffice, it is convenient to add a substantially stoichiometric amount of aqueous potassium hydroxide to a solution of the lactone starting material in a hydrocarbon solvent such as benzene, toluene or cyclohexane containing a small amount of a C.sub.1-3 alkanol, preferably isopropanol, ethanol or methanol, or alternatively in tetrahydrofuran (THF) with or without added alkanol, stirring for a few minutes to about an hour and finally concentrating to dryness in vacuo. The residue is subjected to rigorous drying such as by azeotropic distillation with cyclohexane, toluene or dry tetrahydrofuran, preferably extremely (less than 0.08 mg H.sub.2 O/mL) dry tetrahydrofuran.
The dry alkali metal salt is dissolved in an ethereal solvent such as tetrahydrofuran, diethyl ether, 1,2-dimethoxyethane, cooled to about -80.degree. C. to -25.degree. C., preferably -35.degree. C. to -30.degree. C. and treated with an excess of a strong base such as an alkali metal amide, wherein the alkali metal is lithium, sodium or potassium, preferably lithium, and the amide is diethylamide, pyrrolidide, dimethylamide or diisopropyl amide in an ethereal solvent in a dry, inert environment. After about 2 to 8 hours, preferably about two hours at -80.degree. to -25.degree. C., preferably -35.degree. to -30.degree. C., a methyl halide, such as methyl bromide, methyl chloride or methyl iodide, preferably methyl bromide or methyl iodide, is added to the mixture while maintaining the low temperature. Treatment with the strong base and methyl halide as described can be repeated if appreciable amounts of starting material remain. After 0.5 to about 3 hours following final addition of methyl halide, the reaction mixture is quenched by adding to it excess water.
Following this direct methylation, attempts to convert unreacted lovastatin to the triol acid or the diol lactone for final product purification purposes were made using NaOH or LiOH. However, this base hydrolysis hydrolyzed only a small percentage of the lovastatin. Thus, unreacted lovastatin remained as a contaminant of the final simvastatin product. Furthermore, the base hydrolysis also hydrolyzed simvastatin, thus reducing yields of the desired simvastatin product. Following hydrolysis, the open ring acid form of simvastatin or a salt form thereof was then converted to the lactone by either heat or acid-catalyzed lactonization, and separated and purified by crystallization.